1. Quantification of threonine, serine and tyrosine phosphorylation in TLR signaling pathways. The TLRs are a family of pathogen recognition receptors that alert the host to the presence of pathogens by recognizing molecular signatures, termed pathogen-associated molecular patterns (PAMPs). These sensors act as the first step in the induction of protective innate and adaptive immune responses. There are 11 human TLR homologues and they are each activated by one or more PAMP ligands. TLRs are all transmembrane proteins and their signaling is mediated by association of their internal domains with intracellular components. Classically, the TLR signaling cascade involves the myeloid differentiation primary response gene 88 (MyD88), interleukin-1 receptor-activated kinase (IRAK), and tumor-necrosis factor receptor-associated factor 6 (TRAF6), leading to the activation of Nuclear Factor kappaB (NF-kB). Among the most important genes to be regulated by TLR signaling are those encoding cytokines. Given the key role of cytokines in the orchestration of the inflammatory response, mechanisms of modulating their production has garnered substantial interest, in particular in the area of the development of therapies for the treatment of chronic inflammatory diseases. A clearer understanding of the TLR pathway leading to the cytokine production is required for a successful pharmacological intervention. A) We investigated differences in the phosphoprotein signaling cascades triggered by TLR2, TLR4, and TLR7 ligands using as a responding population a well-characterized murine macrophage cell line. We performed a global, quantitative, early post-stimulation kinetic analysis of the mouse macrophage phosphoproteome using stable isotope labeling with amino acids coupled to phosphopeptide enrichment and high-resolution mass spectrometry. For each TLR ligand, we found marked elevation of phosphorylation of cytoskeleton components, GTPases of the Rho family, and phospholipase C signaling pathway proteins. Phosphorylation of proteins involved in phagocytosis was only seen in response to TLR2 and TLR4 but not to TLR7 activation. Changes in the phosphorylation of proteins involved in endocytosis were delayed in response to TLR2 as compared to TLR4 ligands. These findings reveal that the phosphoproteomic response to stimulation of distinct TLRs varies both in the major modification targets and the phosphorylation dynamics. These results advance our understanding of how macrophages sense and respond to a diverse set of TLR stimuli. The data were deposited in the publicly available online database Proteome Exchange (1). We are characterizing the changes in tyrosine phosphorylation in the same conditions and obtained initial qualitative Western blot data and qualitative mass spectrometry data showing changes in tyrosine phosphorylation following LPS stimulation. We are studying the changes in phosphorylation-dependent signaling in cells where the TLR signaling pathway components (MyD88, IRAK family proteins, CD14) are knocked down or knocked out. We will use the phosphorylation datasets as well as planned datasets quantifying other PTMs (ADP-ribosylation, GlcNAc, ubiquitination) as additional constraints for a computational model of the TLR signaling network (project AI001085-07: Absolute Quantification of Molecular Representation and Interaction). The candidate proteins whose phosphorylation changed significantly during the investigated time course are being further examined in follow up biological experiments. We have characterized in detail the changes in phosphorylation of specific sites of MARCKS upon LPS stimulation and we are now exploring the biological significance of these sites. We have performed site-directed mutagenesis of the individual sites and the mutant MARCKS is being expressed in the cells where we knocked out the wild-type protein using CRISPR technology. B). We have optimized another relative quantification method based on post-processing peptide labeling - the dimethylation method, which should be extremely useful for proteomic and phosphoproteomic studies of primary cells and tissues not amenable to the SILAC protocol. The method was tested on murine macrophage lysates and the results were comparable to the SILAC- quantified study. We have applied dimethyl labeling to studies of the TLR phosphoproteome in primary murine macrophages and human elutriated monocytes. C). To complete the TLR signaling network and assess the proteome dynamics, we have conducted parallel studies of the proteome and secretome changes using the same cells and ligands as for the phosphoproteome analysis, but collecting data after longer periods of time to allow for changes in protein expression and secretion (2). We have validated the data using ELISA-based assays of cytokine production. We have performed data correlation with the transcriptome (in collaboration with Iain Fraser). The data will provide more stringent constraints for the TLR signaling model. 2. Analysis of post-translational modifications of CENP-A (collaboration with Yamini Dalal, NCI): In eukaryotes DNA is packaged into chromatin by essential histone proteins. Specialized histone variants such as centromere-specific histone H3 (CENP-A) provide a structural and epigenetic basis for chromosome segregation by marking centromeres. To maintain centromere parity after replication, CENP-A must segregate equally to nascent daughter DNA strands. How cells prevent unequal distribution of CENP-A to daughter strands after replication fork passage is unknown. We have characterized novel modifications within the histone fold domains of CENP-A and H4 that occur at G1-S cell cycle transition, which coincide with loss of the chaperone HJURP binding, suggesting a mechanistic basis for CENP-A structural conversion. We have established that accurate post-translational modification mapping is possible from minute amounts of protein, using ultra-sensitive mass spectrometry, and bioinformatic tools combined with careful manual spectra validation. We are continuing the detailed characterization of the modification states of CENP-A dependent on the cell cycle stage. Time-resolved, stimulus-dependent quantitative changes of phosphorylation of individual sites are being obtained using sensitive nanospray-based mass spectrometry (LTQ Orbitrap Velos from Thermo) combined with Single Reaction Monitoring. We are now using TAU gels for the optimal separation of histone variants to obtain comprehensive modification map of CENP-A in the primary tumor cells. The TAU gels and mass spectrometry are a novel combination of methods for histone post-translational modification analysis (3). 3. We have performed a quantitative analysis of the proteome of the cells from the terminal ileum (chosen as a site of intense host-microbe interactions) of germ-free and normal mice (collaboration with Drs. Natalia Shulzhenko and Andrey Morgun from Oregon Sate University). 39 mice were used in the study. The samples were chemically labeled with iTRAQ and analyzed on the Orbitrap Velos mass spectrometer. The data showed large changes in the immune processes-related protein expression as well as in certain metabolic pathways. The correlation of the proteome and transcriptome data revealed several differentially regulated pathways. References: 1. Sjoelund V, Smelkinson M, and Nita-Lazar A. (2014) J Proteome Res. 2014 Nov 7;13(11):5185-97. doi: 10.1021/pr5002466. 2. Koppenol-Raab M., and Nita-Lazar A. Methods in Molecular Biology, in press. 3. Nuccio A., Bui M., Dalal Y., and Nita-Lazar A. Methods in Enzymology 586, in press.